Supercritical CO₂-Based Extraction and Detection of Phenolic Compounds and Saponins from the Leaves of Three Medicago varia Mart. Varieties by Tandem Mass Spectrometry

Abstract

A comparative metabolomic study of three varieties of alfalfa (Medicago varia Mart.) was performed via extraction with supercritical carbon dioxide modified with ethanol (EtOH) and the detection of bioactive compounds via tandem mass spectrometry. Several experimental conditions were investigated in the pressure range of 50–250 bar, with ethanol used as a co-solvent in an amount of 1% of the total volume in the liquid phase at a temperature in the range of 31–70 °C. The most effective extraction conditions were as follows: a pressure of 250 Bar and a temperature of 60 °C for M. varia. M. varia contains various phenolic compounds and sulfated polyphenols with valuable biological activity. Tandem mass spectrometry (HPLC-ESI–ion trap) was applied to detect the target analytes. A total of 103 bioactive compounds (59 polyphenols and 44 compounds belonging to other chemical groups) were tentatively identified in extracts from aerial parts of alfalfa. For the first time, twenty-one chemical constituents from the polyphenol group (flavones: Formononetin, Chrysoeriol, Cirsimaritin, Cirsiliol, Cirsilineol, tricin-O-hexoside, Apigenin C-glucose C-deoxyhexoside, Apigenin 7-O-diglucuronide, 2′-Hydroxygenistein 4′,7-O-diglucoside, etc.) and six from other chemical groups (saponins: Soyasaponin II, Soyasaponin gamma g, Soyasaponin I, Soyasaponin Bd, Soyaysaponin beta g, etc.) were identified in the aerial parts of M. varia.

1. Introduction

One of the most versatile and cost-effective crops is alfalfa (Medicago varia Mart., family Fabaceae Lindl.), which plays an important role not only in sustainable agriculture but also in expanding the raw material base of the food, cosmetic and pharmaceutical industries. For instance, studies have shown that Medicago varieties contain bioactive compounds, identified as phenolic compounds, in particular, flavonoids. The flavonoids found include flavones (luteolin and apigenin), isoflavones (genistein), flavanones (naringenin), flavanols (catechin and epicatechin) and anthocyanidins (cyanidin and delphinidin) [1,2]. The interest in flavonoid research increased because of the potential of these substances to prevent or treat factors related to metabolic disorders [3]. GC–MS analysis of M. sativa seeds revealed their enrichment in crude protein (33.79%), crude oil (8.11%), squalene, hexadecanoic acid methyl ester, n-hexadecanoic acid, 9,12-octadecadienoic acid methyl ester, 9-octadecenamide and vitamin E. Moreover, Medicago sativa seed inclusion in the diet is recommended to normalize serum cholesterol levels in type II hyperlipoproteinemia patients [4,5,6]. Additionally, there are several interesting scientific studies showing the presence of simple phenolic compounds in Medicago [7,8]. These compounds have a range of biological activities, including anti-inflammatory, antioxidant, phytoestrogenic and anticarcinogenic abilities, as well as their interactions with intracellular signaling pathways and regulation of cell survival. Extracts and balms of M. sativa have long been used as traditional herbal medicines in many countries, such as China, India and America [9,10]. Most varieties of alfalfa are autotetraploids (2n = 4x = 32), with the main number of chromosomes being eight [11,12]. Alfalfa is characterized by its exceptional ability to grow under a wide range of natural conditions, its stable global yield, and its longevity and reproduction of soil fertility through fixation of atmospheric nitrogen. This crop is used as fodder in its green form or for the preparation of fodder (hay, haylage and grass meal). Lucerne hay is a quality forage containing high levels of protein, phosphorus, calcium and essential amino acids [13]. Increased forage production is possible through the development of more productive and higher-quality lucerne varieties. Each soil–climatic zone requires a diverse set of complementary varieties adapted to different extreme growing conditions [14]. Therefore, the study of the genetic diversity of alfalfa source material is of great theoretical and practical importance. M. varia contains omega-3 fatty acids, which are necessary to improve milk quality and increase meat production in ruminants [15]. The plant complex of alfalfa, which contains substances necessary for humans (especially in unfavorable environmental conditions), is used in technology for the production of fermented milk products [16]. It is also worth noting the insecticidal and fungicidal potential of the use of saponins identified in Medicago. The combined deterrent and toxic effects on insects make Medicago saponins suitable for use against insect pests in agriculture and horticulture [17]. An effective way of using Medicago saponins against insect herbivores is to select varieties that accumulate high levels of saponins [17]. For example, the development, survival and reproduction of pea aphids fed on high-saponin alfalfa were reduced compared to those fed on low-saponin alfalfa. In addition, it has been shown that zanhic acid tridesmoside and medicagenic acid, which accumulate in a high-saponin cultivar, are the main compounds contributing to the resistance of alfalfa to pea aphids [18,19]. The nematocidal activity of saponins allows the use of Medicago biomass as a biological agent to control plant-parasitic nematodes, which are widespread in the soil. The antifungal activity of alfalfa saponins may also reduce the presence of phytopathogenic fungi in the soil. Total saponins and selected compounds from different Medicago species have been shown to prevent fusarium on tulip bulbs [20,21]. Saponins against phytopathogenic fungi and nematodes in plant material make Medicago biomass particularly useful as an agent against soil-borne plant pathogens and as a biological fertilizer. Supercritical fluid extraction with the use of pressured CO₂ (SC-CO₂) has been used over the last 50 years in analytical methodologies to investigate the composition of food products, for the removal of undesirable substances and for the isolation of valuable molecules. The goal of the present work was to identify and select bioactive compounds from M. varia via extraction with SC-CO₂. Also, a tandem mass spectrometry protocol was used for the detailed screening of phytochemicals present in three varieties of M. varia.

2. Materials and Methods

2.1. Materials

The subject of the study was the green mass of M. varia varieties (Demetra, Nakhodka and Sarga) (Figure 1A–D) collected and grown at the Sakhalin Agricultural Scientific Research Institute—Branch of N.I. Vavilov All-Russian Institute of Plant Genetic Resources. Standard agronomic practices were used for growing the accessions/varieties in their respective locations. The aerial parts of M. varia were harvested at the end of July 2023. All plant tissues used in this work conformed to the standard established by the State Pharmacopoeia of the Russian Federation [22].

2.2. Chemicals and Reagents

All reagents used in the study were of analytical grade. HPLC-grade acetonitrile was purchased from Fisher Scientific (Kent, UK), and MS-grade formic acid and ethanol (EtOH) were purchased from Sigma-Aldrich (Steinheim, Germany). Ultrapure water was obtained from Siemens (SIEMENS water technologies, Munich, Germany).

2.3. Extraction

SC-CO₂ extraction was performed using the SFE-500 supercritical pressure extraction system (Thar SCF Waters, Milford, CT, USA). The system options include the following: a co-solvent pump (Thar Waters P-50 High Pressure Pump) for the extraction of polar samples; a CO₂-flow meter (Siemens, Munich, Germany) to measure the amount of CO₂ supplied to the system; and multiple extraction vessels to extract different sample sizes or to increase the throughput of the system. The flow rate was 10–25 mL/min for liquid CO₂ and 1.00 mL/min for EtOH. Extraction samples of 200 g M. varia were used. The extraction time was counted after reaching working pressure and equilibrium flow and was 60–90 min for each sample. This method of SC-CO₂ extraction of plant matrices was tested by the authors on numerous plant samples, including aboveground and underground parts of the plant [23,24,25].

2.4. Liquid Chromatography

High-performance liquid chromatography was carried out on a Shimadzu LC-20 Prominence HPLC (Shimadzu, Kyoto, Japan) instrument equipped with a UV sensor and a C18 silica reverse phase column (4.6 × 150 mm, particle size: 2.7 μm). Mobile-phase eluent A was deionized water containing 0.1% formic acid, and eluent B was acetonitrile containing 0.1% formic acid. The gradient elution was started at 0–2 min, 0% eluent B 2–50 min, 0–100% B; control washing: 50–60 min, 100% B. The mobile-phase flow rate and column temperature were maintained at 0.3 mL/min and 30 °C, respectively. A UV–vis detector, the SPD-20A (Shimadzu, Kyoto, Japan), was used for detection and compound identification at a wavelength of 230 nm. The injection volume was 10 µL. Additionally, liquid chromatography was combined with a mass spectrometric ion trap to identify compounds.

2.5. Mass Spectrometry

MS analysis was performed on an ion trap, the amaZon SL (Bruker Daltoniks, Bremen, Germany), equipped with an ESI source in negative ion mode. MS analysis was carried out in electrospray ionization (ESI) mode using negative and positive polarity for all samples with data-independent MSE acquisition. The optimized parameters were obtained as reported earlier [23,24,25]. Similarly, the data collection and compound identification were carried out as per our previous reports [23,24,25].

2.6. Statistical Analysis

To more clearly present the similarities and differences of bioactive substances identified in different variants of M. varia, the team of authors used the Jaccard index. The Jaccard index, also known as the Jaccard similarity coefficient, is a statistical measure used to evaluate the similarity and diversity of sets of samples. Nine replicate samples were analyzed. Jaccard indices were calculated using a the “Compare Lists—Multiple List Comparator” hosted on molbiotools server (https://molbiotools.com/listcompare.php (accessed on 21 March 2024)).

3. Results

3.1. SC-CO₂ Extraction of Aerial Parts of M. varia

Three M. varia varieties, i.e., Sarga, Nakhodka and Demetra, were examined by SC-CO₂ extraction under different extraction conditions. The supercritical pressures applied ranged from 50 to 250 bar, and the extraction temperature ranged from 31 to 70 °C. The co-solvent, EtOH, was used in an amount of 1% of the total solvent amount. The

Table 1. The global yield of extract (mg/100 mg) after SC-CO₂ extraction of M. varia, variety Sarga.

Temperature (°C)	Pressure (Bar)
	50 Bar	100 Bar	150 Bar	200 Bar	250 Bar
31 °C	0.4	1.2	1.9	1.8	1.8
40 °C	0.7	1.9	2.1	1.9	1.9
45 °C	1.2	2.7	2	2	2.1
50 °C	1.3	2.5	3.1	2.5	2.2
55 °C	1.4	2	3.1	2.6	2.1
60 °C	2.5	2.9	2.5	2.4	2.1
70 °C	2.3	2.9	2.2	2.2	1.9

shows the global yield of bioactive compounds (variety Sarga) by SC-CO₂ extraction. Figure 1D shows a 3D plot of the global yield of bioactive compounds during SC-CO₂ extraction of the aerial parts (variety Sarga).

The maximum global yield of bioactive substances from alfalfa aerial parts (variety Sarga) was observed under the following extraction conditions:

–

Pressure: 150 Bar, extraction temperature: 50 °C, extraction time: 1 h; the global yield of biologically active substances was 3.1 mg/100 mg of plant sample; the share of the EtOH modifier was 2%;

–

Pressure: 150 Bar, extraction temperature: 55 °C, extraction time: 1 h; the global yield of biologically active substances was 3.1 mg/100 mg of plant sample; the share of the EtOH modifier was 2%.

Table 2. The global yield of bioactive compounds (variety Nakhodka) by SC-CO₂ extraction.

Pressure	50 Bar	100 Bar	150 Bar	200 Bar	250 Bar
31 °C	0.4	1.2	1.6	1.7	1.7
40 °C	0.7	1.9	2.3	1.9	1.9
45 °C	1.2	2.7	2	2	2.1
50 °C	1.3	2.5	2.7	2.2	2.2
55 °C	1.4	2	2.8	2.4	2.1
60 °C	2.5	2	2.5	2.4	2.1
70 °C	2.3	2.4	2.2	2.2	1.9

showing the global yield of bioactive compounds (variety Nakhodka) by SC-CO₂ extraction is presented below.

Figure 1E shows a 3D graph of the global yield of biologically active substances during SC-CO₂ extraction of alfalfa aerial parts (variety Nakhodka). The maximum global yield of bioactive substances from alfalfa aerial parts (variety Nakhodka) was observed under the following extraction conditions:

–

Pressure: 150 Bar, extraction temperature: 50 °C, extraction time: 1 h; the global yield of biologically active substances was 2.7 mg/100 mg of plant sample; the share of the EtOH modifier was 2%;

–

Pressure: 150 Bar, extraction temperature: 55 °C, extraction time: 1 h; the global yield of biologically active substances was 2.8 mg/100 mg of plant sample; the share of the EtOH modifier was 2%.

Table 3. The global yield of bioactive compounds (variety Demetra) by SC-CO₂ extraction.

Pressure	50 Bar	100 Bar	150 Bar	200 Bar	250 Bar
31 °C	0.4	1.2	1.6	1.7	1.7
40 °C	0.7	1.9	2.3	1.9	1.9
45 °C	1.2	2.7	2	2	2.1
50 °C	1.3	2.5	2.7	2.2	2.2
55 °C	1.4	2	3.2	2.4	2.1
60 °C	2.5	2	2.5	2.4	2.1
70 °C	2.3	2.4	2.2	2.2	1.9

shows the global yield of bioactive compounds (variety Demetra) by SC-CO₂ extraction.

Figure 1F shows a 3D graph of the global yield of biologically active substances during SC-CO₂ extraction of alfalfa aerial parts (variety Demetra). The maximum global yield of bioactive substances from alfalfa aerial parts (variety Demetra) was observed under the following extraction conditions:

–

Pressure: 150 Bar, extraction temperature: 50 °C, extraction time: 1 h; the global yield of biologically active substances was 3.2 mg/100 mg of plant sample; the share of the EtOH modifier was 2%.

3.2. Global Metabolome Profile of M. varia

The structural identification of each compound was carried out on the basis of its accurate mass and MS/MS fragmentation by HPLC-ESI–ion trap–MS/MS. A total of 103 chemical compounds were identified from the extracts of the three M. varia varieties (

Table 4. Jaccard indices for three varieties of M. varia.

	Variety Demetra (71)	Variety Nakhodka (63)	Variety Sarga (38)
Variety Demetra (71)	--	41 0.4409	25 0.2976
Variety Nakhodka (63)	41 0.4409	--	22 0.2785
Variety Sarga (38)	25 0.2976	22 0.2785	--

). Fifty-nine and forty-four chemical compounds were classified as polyphenols and others, respectively (see the chemical structure of some of these compounds in Figure 2). The polyphenols detected in our study were categorized as flavones, flavonols, flavan-3-ols, anthocyanidins, phenolic acids, lignans, coumarins, stilbenes, etc. In total, the metabolites detected in our study belonged to 19 compound classes. The highest number of metabolites was recorded for flavones (24), followed by flavonols (20), anthocyanins (6) and flavan-3-ols (3). These numbers of compounds in respective groups indicate that M. varia extracts are rich in flavonoids. The highest numbers of chemical compounds from other groups were recorded for polysaccharides (8) and saponins (11).

Figure 3 shows the numbers of common and specific compounds. Nineteen compounds were commonly detected from the three M. varia varieties. These nineteen compounds belong to compound classes such as flavones, flavonols, anthocyanins and saponins, suggesting that flavonoids and saponins are major active compounds in M. varia leaves. The applied methods were able to detect 71 (Demetra), 63 (Nakhodka) and 38 (Sarga) compounds.

Moreover, to present the similarities and differences in bioactive substances in different variations of M. varia, we used the Jaccard index (Table 4). The Jaccard index, also known as the Jaccard similarity coefficient, is a statistic used to evaluate the similarity and diversity of sets of samples [26,27,28]. It showed that the highest degree of similarity existed between the varieties Demetra and Nakhodka—0.4409.

3.2.1. Flavones

Hydroxy(iso)flavones

7-hydroxyisoflavone formononetin (compound 1) and monohydroxy flavone apigenin-7,4′-dimethyl ether (compound 6) have already been reported in Astragali Radix [29], Huolisu oral liquid [30], Dracocephalum jacutense [31], Maackia amurense [32], the Chinese herbal formula for the Jian-Pi-Yi-Shen pill [33], Ocimum [34] and propolis [35]. Thus, our results indicate that both the flavones formononetin and apigenin-7,4′-dimethyl ether were tentatively identified components in the extracts from M. varia (varieties Demetra and Nakhodka) (Table 2). The CID (collision-induced spectrum) in positive ion mode of flavone formononetin from variety Demetra is shown in Figure 4A. [M + H]⁺ ions produced two fragment ions (FIs) with m/z 254.12 and m/z 213.20 (Figure 4A). The FI with m/z 254.12 produced one characteristic daughter ion with m/z 237.13.

Dihydroxyflavones

The flavones acacetin (compound 3), dihydroxy-methoxy(iso)flavone (compound 4), cirsimaritin (compound 9), dihydroxy-dimethoxy(iso)flavone (compound 10), nevadensin (compound 12), cirsilineol (compound 13) and 5,6-dihydroxy-7,8,3′,4′-tetramethoxyflavone (compound 15) (Table 2) have already been characterized as components of Mentha [36], Ocimum [34], Mexican lupine species [37], Wissadula periplocifolia [38], Artemisia annua [39], Rosmarinus officinalis [40], Astragali radix [29] and propolis [35]. We also tentatively identified these flavones in extracts from the three varieties of M. varia (Nakhodka, Sarga and Demetra). The CID in positive ion mode of cirsimaritin from extracts from M. varia (variety Nakhodka) is shown in Figure 4B. [M + H]⁺ ions produced four FIs with m/z 287.18, m/z 259.20, m/z 216.08 and m/z 167.17 (Figure 4B). The FI with m/z 259.20 produced two characteristic daughter ions with m/z 227.15 and m/z 171.18. The FI with m/z 227.15 generated an ion with m/z 198.18.

Trihydroxyflavones

The flavones apigenin (compound 2), trihydroxymethoxyflavone (compound 7), chrysoeriol [chrysoeriol] (compound 8), cirsiliol (compound 11), luteolin 7-O-glucoside [cynaroside] (compound 16), kaempferide [4′-O-methylkaempferol] (compound 26), rhamnocitrin (compound 27), kaempferol-3-O-α-L-rhamnoside (compound 33), astragalin [kaempferol 3-O-glucoside] (compound 34) and isorhamnetin 3-O-glucoside (compound 36) (Table 2) have been already characterized as a components of Inula gaveolens [41], Phlomis (Lamiaceae) [42], Lonicera henryi [43], Ribes meyeri [44], Lonicera japonica [45], Stevia rebaudiana [46], propolis [35] and Jatropha [47]. There trihydroxyflavones were tentatively identified in extracts from M. varia (varieties Nakhodka, Sarga and Demetra). The CID in negative ion mode of astragalin from extracts of M. varia is shown in Figure 4C. [M − H]⁻ ions produced four FIs with m/z 285.20, m/z 401.15, m/z 327.21 and m/z 255.18 (Figure 4C). The FI with m/z 285.20 produced one characteristic daughter ion with m/z 255.19, m/z 227.15 and m/z 151.250. The FI with m/z 255.19 produced one characteristic daughter ion with m/z 227.22. Astragalin has been reported in extracts from Juglans mandshurica [48], bee pollen [49], Lonicera japonica [45], Ribes meyeri [44] and potato [50].

3.2.2. Flavan-3-ols

Catechin (compound 45), (epi)-catechin (compound 46) and gallocatechin (compound 47) (Table 2) have already been characterized as components of Carpinus betulus [51], Solanaceae [52], Glottiphyllum linguiforme [53], Embelia [54], Inula viscosa [55], Juglans mandshurica [48], Ribes meyeri [44], Radix polygoni multiflori [56], Glycine soja [57,58] and Jatropha [47]. These flavan-3-ols were tentatively identified in extracts from M. varia (varieties Nakhodka, Sarga and Demetra). The CID in positive ion mode of Catechin from extracts from M. varia (variety Sarga) is shown in Figure 4D. [M − H]⁻ ions produced five FIs with m/z 273.18, m/z 245.17, m/z 229.23, m/z 188.17 and m/z 130.23 (Figure 4D). The FI with m/z 273.18 produced four characteristic daughter ions with m/z 245.16, m/z 227.21, m/z 184.17 and m/z 130.27. The FI with m/z 245.16 produced three characteristic daughter ions with m/z 227.20, m/z 201.19 and m/z 159.30.

3.2.3. Anthocyanins

Cyanidin-3-O-glucoside (compound 48), malvidin 3-O-glucoside (compound 49), cyanidin 3-(6″-malonylglucoside) (compound 50), cyanidin-3-O-dioxayl-glucoside (compound 51), peonidin 3-O-(6-O-p-coumaroyl) glucoside (compound 52) and malvidin 3-O-(6-O-p-caffeoyl) glucoside (compound 53) (Table 2) have been already characterized as components of Gaultheria mucronata, Gaultheria antarctica [59], Berberis microphylla [60], Bougainvillea [61], Grape [62], vines [63] and many other plant species whose organs (mainly fruits) accumulate pigments and exhibit a range of colors. These anthocyanins were tentatively identified in extracts from M. varia (varieties Nakhodka and Demetra). The CID in positive ion mode of malvidin 3-O-(6-O-p-caffeoyl) glucoside from extracts from M. varia (variety Nakhodka) is shown in Figure 4E. [M + H]⁺ ions produced two FIs with m/z 331.17 and m/z 257.05 (Figure 4E). The FI with m/z 331.11 produced three characteristic daughter ions with m/z 315.11, m/z 270.11 and m/z 153.11. The anthocyanin malvidin 3-O-(6-O-p-caffeoyl) glucoside was tentatively identified in results for extracts from Grape [62] and Bougainvillea [61]. The CID in positive ion mode of cyanidin-3-O-glucoside from extracts from M. varia (variety Demetra) is shown in Figure 4F. [M + H]⁺ ions produced one FI with m/z 287.13 (Figure 4F). The FI with m/z 287.13 produced two characteristic daughter ions with m/z 241.09 and m/z 165.10. The anthocyanin cyanidin-3-O-glucoside was tentatively identified in the results for extracts from several plant species, some of which were mentioned at the beginning of this paragraph, as well as others, such as Ribes magellanicum [64] and Rubus ulmifolius [65].

3.3. Newly Detected Chemical Compounds in M. varia

Of the detected metabolites in the three M. varia varieties, twenty-one compounds from the polyphenol group and six compounds from other chemical groups were identified for the first time. The newly identified polyphenols include flavones (formononetin, chrysoeriol, cirsimaritin, cirsiliol, cirsilineol, tricin-O-hexoside, apigenin C-glucose C-deoxyhexoside, apigenin 7-O-diglucuronide and 2′-hydroxygenistein 4′,7-O-diglucoside malonylated), flavonols (ampelopsin, astragalin, kaempferol 3-(6″-malonylglucoside), rhamnosylhexosyl-methyl-quercetin, quercetin 3,4′-di-O-β-glucopyranoside, isorhamnetin-di-O-hexoside, quercetin-7-O-(acetyl-hexoside)-3-O-rhamnoside and isorhamnetin-3-O-6-O-acetyl-β-D-glucopyranosyl), the flavan-3-ol gallocatechin, vimalin (phenylpropanoid), syringaresinol (lignan), fraxetin (coumarin), etc. Interestingly, the other compound classes that we also detected in M. varia were saponins (soyasaponin II, soyasaponin gamma g, soyasaponin I, soyasaponin Bd and soyasaponin beta g), steroidal alkaloids (alpha-chaconine), etc. (

Table 5. Chemical compounds identified from the SC-CO₂-extracts of M. varia in positive and negative ionization modes by HPLC–ion trap–MS/MS.

	Class of Compound	Identification	Formula	Calculated Mass	Observed Mass [M − H]−	Observed Mass [M + H]+	MS/MS Stage 1 Fragmentation	MS/MS Stage 2 Fragmentation	MS/MS Stage 3 Fragmentation	References
		Phenolic compounds
1	7-Hydroxyisoflavone	Formononetin [Biochanin B; Formononetol] *	C16H12O4	268.2641		269	254; 213	237	237	Astragali Radix [29]; Huolisu oral liquid [30]; Dracocephalum jacutense [31]; Maackia amurense [32]; Chinese herbal formula, Jian-Pi-Yi-Shen pill [33]
2	Flavone	Apigenin [5,7-Dixydroxy-2-(40Hydroxyphenyl)-4H-Chromen-4-One]	C15H10O5	270.2369		271	271; 153			Propolis [35]; Inula gaveolens [41]; Phlomis (Lamiaceae) [42]; Lonicera henryi [43]; Ribes meyeri [44]; Lonicera japonica [45]; Stevia rebaudiana [46]; Jatropha [47]
3	Flavone	Acacetin [Linarigenin; Buddleoflavonol]	C16H12O5	284.2635		285	270	269; 242	213; 185	Propolis [35]; Mentha [36]; Mexican lupine species [37]; Wissadula periplocifolia [38]
4	Flavone	Dihydroxy-methoxy(iso)flavone	C16H12O5	284.2635		285	270	269	227	Propolis [35]
5	Flavone	Luteolin	C15H10O6	286.2363		287	213; 165	157		Propolis [35]; Artemisia absinthium [39]; Inula gaveolens [41]; Lonicera henryl [43]; Ribes meyeri [44]; Lonicera japonica [45]; Jatropha [47]; potato [50]
6	Flavone	Apigenin-7, 4′-dimethyl ether	C17H14O5	298.2901		299	284	256; 169	255; 132	Ocimum [34]; propolis [35]
7	Flavone	Trihydroxymethoxyflavone	C16H12O6	300.2629		301	286	258		Artemisia absinthium [39]
8	Flavone	Chrysoeriol [Chryseriol] *	C16H12O6	300.2629		301	286	258		Propolis [35]; Mentha [36]; Mexican lupine species [37]; Rhus coriaria [66]
9	Flavone	Cirsimaritin [Scrophulein; 4′,5-Dihydroxy-6,7-Dimethoxyflavone] *	C17H14O6	314.2895		315	287; 259; 216	227; 171	198	Ocimum [34]; Artemisia annua [39]; Rosmarinus officinalis [40]
10	Flavone	Dihydroxy-dimethoxy(iso)flavone	C17H14O6	314.2895		315	287; 259; 216	227; 171	198	Astragali radix [29]; propolis [35]; Rosmarinus officinalis [50]
11	Flavone	Cirsiliol *	C17H14O7	330.2889		332	315; 271			Ocimum [34]; Juglans mandshurica [48]; Inula viscosa [55]
12	Flavone	Nevadensin	C18H16O7	344.3154		345	312; 222; 181	284; 256	283; 269; 255	Ocimum [34]; Mentha [36]
13	Flavone	Cirsilineol [Eupatrin; Fastigenin] *	C18H16O7	344.3154		345	312; 222; 181	284; 256	283; 269; 255	Ocimum [34]
14	Flavone	Tetrahydroxy-dimethoxyflavone	C17H14O8	346.2883	345		330	315	287	Artemisia absinthium [39]
15	Flavone	5,6-Dihydroxy-7,8,3’,4’-tetramethoxyflavone	C19H18O8	374.3414		375	368; 348; 325; 304	358; 301; 226		Mentha [36]
16	Flavone	Luteolin 7-O-glucoside [Cynaroside; Luteoloside]	C21H20O11	448.3769		449	287	241; 165	213; 147	Mexican lupine species [37]; Lonicera henryi [43]; Lonicera japonica [45]
17	Flavone	Luteolin 8-C-Glucoside [Orientin; Orientin (Flavone); Lutexin]	C21H20O11	448.3769		449	430; 373; 328; 285; 215			Lemon, passion fruit [67]; P. aculeata [68]; Phyllostachys nigra [69]; Aspalathus linearis [70]; bamboo [71]
18	Flavone	Tricin O-hexoside *	C23H24O12	492.4295		493	331	315		Triticum aestivum L. [72]; bamboo [71]
19	Flavone	Dihydroxy-trimethoxyflavone-O-hexoside	C23H22O14	506.414		507	331	315; 270	270	Citrus species [73]
20	Flavone	Apigenin O-pentosyl hexoside	C26H28O14	564.4921		565	433; 288	415; 334; 271; 163	127	F. glaucescens [53]
21	Flavone	Apigenin C-glucose C-deoxyhexoside *	C27H30O14	578.5187		579	547; 488; 403; 365			Passiflora incarnata [74]
22	Flavone	Apigenin 7-O-diglucuronide *	C27H26O17	622.4851		623	447	271	153	Perilla frutescens [75]
23	Flavone	Tricin di-O,O-hexoside	C29H34O17	654.5701		655	331; 493	315; 270; 153	270	Triticum aestivum L. [72,76]
24	Flavone	2′-Hydroxygenistein 4′, 7-O-diglucoside malonylated *	C30H32O19	696.5637		697	287	241; 165; 121	185	Mexican lupine species [37]
25	Flavonol	Kaempferol	C15H10O6	286.2363		287	241	165		Ribes meyeri [44]; Lonicera japonicum [45]; Juglans mandshurica [48]; Rhus coriaria [66]
26	Flavonol	Kaempferide [4′-O-Methylkaempferol]	C16H12O6	300.2629		301	286	258		Ribes meyeri [44]; Alpinia officinarum [77]; Brazilian propolis [78]
27	Flavonol	Rhamnocitrin	C16H12O6	300.2629		301	273; 163	243	227	Astragali radix [29]; Lonicera caerulea [79]
28	Flavonol	Quercetin	C15H10O7	302.2357		303	285; 167	257; 197	257	Inula gaveolens [41]; Juglans mandshurica [48]; Inula viscosa [55]; Black soja [57]
29	Flavonol	Dihydroquercetin (Taxifolin; Taxifoliol)	C15H12O7	304.2516		305	285; 211; 175	268; 185; 124	168	Juglans mandshurica [48]; Glycine soja [58]; Camellia kucha [80]
30	Flavonol	Isorhamnetin [Isorhamnetol; Quercetin 3′-Methyl ether; 3-Methylquercetin]	C16H12O7	316.2623		317	302	274; 153	229; 153	Propolis [35]; Rosmarinus officinalis [40]; Stevia rebaudiana [46]; Inula viscosa [55]; Lonicera caerulea [79]; Phoenix dactylifera [81]
31	Flavonol	Myricetin	C15H10O8	318.2351	317		273; 295	260	238	Juglans mandshurica [48]; F. glaucescens [53]; Taraxacum officinale [82]
32	Flavonol	Ampelopsin [Dihydromyricetin; Ampeloptin] *	C15H12O8	320.251		321	301; 201	267; 201		Juglans mandshurica [48]; Rnus coriaria [66]; Impatients glandulifera Royle [83]
33	Flavonol	Kaempferol-3-O-α-L-rhamnoside	C21H20O10	432.3775		433	427; 340; 287			Carpinus betulus [51]; C. edulis; F. glaucescens [53]; Taraxacum officinale [82]; Cassia abbreviata [84]
34	Flavonol	Astragalin [Kaempferol 3-O-glucoside] *	C21H20O11	448.3769	447		285; 255	255; 227; 151	227	Juglans mandshurica [48]; bee pollen [49]; Lonicera japonica [45]; Ribes meyeri [44]; potato [50]
35	Flavonol	Quercetin 3-O-glucoside [Isoquercetin; Isoquercitrin; Hirsutrin; Quercetin-3-O-Glucopyranoside]	C21H20O12	464.3763		465	303; 258; 164	243; 179		Juglans mandshurica [48]; Black soja [57]; Ribes meyeri [44]; Lonicera henryi [43]; Lonicera japonica [45]; Vaccinium myrtillus [85]; Solanaceae [52]; Rhus coriaria [66]; Embelia [54]
36	Flavonol	Isorhamnetin 3-O-glucoside	C22H22O12	478.4029		479	317	301; 257; 177	274; 218	Artemisia annua [86]; Eucalyptus [87]; Capsicum annuum [88]; Senecio clivicolus [89]
37	Flavonol	Isorhamnetin 3-O-glucoronide	C22H20O13	492.3864		493	317	302	274	Anethum graveolens [90]; Strawberry [91];
38	Flavonol	Kaempferol 3-(6″-malonylglucoside) *	C24H22O14	534.4231		535	287	241; 165	213	A. cordifolia [53]; Strawberry [91]
39	Flavonol	Rhamnosylhexosyl-methyl-quercetin *	C26H28O17	612.4903		613	595; 540; 489	521; 337; 241	503; 349; 239	Phoenix dactylifera [81]
40	Flavonol	Quercetin 3,4’-di-O-beta-glucopyranoside [Quercetin diglucoside] *	C27H30O17	626.5169		627	465; 393; 303	303	257; 165	Potato leaves [92]; potato [50]; rapeseed petals [93]
41	Flavonol	Quercetin-O-dihexoside	C27H30O17	626.5169		627	465; 393; 303	303	257; 165	Inula viscosa [55]; Artemisia absinthium [39]; Chilean currants [64]; Phoenix dactylifera [81]; Taraxacum formosanum [94]
42	Flavonol	Isorhamnetin-di-O-hexoside [Methyl quercetin-O-dihexoside] *	C28H32O17	640.5435		641	317	302	285; 228; 169	Artemisia absinthium [39]; passion fruit [67]; Phoenix dactylifera [81]
43	Flavonol	Quercetin-7-O-(acetyl-hexoside)-3-O-rhamnoside *	C29H32O17	652.5542		653	301	286; 153	258	Capsicum annuum [88]
44	Flavonol	Isorhamnetin-3-O-6-O-acetyl-beta-D-glucopyranosyl *	C30H34O18	682.5802		683	331	315; 270		Rosa rugosa [95]
45	Flavan-3-ol	Catechin	C15H14O6	290.2681		291	273; 188	245	227; 201	Inula viscosa [55]; Juglans mandshurica [48]; Black soja [57]; Glycine soja [58]
46	Flavan-3-ol	(epi)-Catechin	C15H14O6	290.2681		291	261; 173	173		Black soja [57]; Glycine soja [58]; Jatropha [47]
47	Flavan-3-ol	Gallocatechin [+(-)Gallocatechin] *	C15H14O7	306.2675		307	289	260; 175	244; 171	Carpinus betulus [51]; Solanaceae [52]; G. linguiforme [53]; Ribes meyeri [44]; Embelia [54]
48	Anthocyanin	Cyanidin-3-O-glucoside [Cyanidin 3-O-beta-D-Glucoside; Kuromarin]	C21H21O11+	449.3848		449	287	241; 165	213; 147	Black soybean [57]; Glycine soja [58]; Ribes magellanicum [64]; Rubus ulmifolius [65]; B. ilicifolia; B. empetrifolia; R. maellanicum; R. cucullatum; M. nummalaria; G. mucronata; G. antarctica; Fuchsia magellanica [59]; B. microphylla [60]
49	Anthocyanin	Malvidin 3-O-glucoside [Oenin]	C23H25O12	493.4374		493	331	315; 270	315; 270	G. mucronata; G. antarctica [59]; Berberis microphylla [60]
50	Anthocyanin	Cyanidin 3-(6″-malonylglucoside)	C24H23O14	535.4310		535	287	241; 165	213	Strawberry [91]; Zostera marina [25]
51	Anthocyanin	Cyanidin-3-O-dioxayl-glucoside	C31H28O12	592.5468		592	287	241	227; 209; 144	Rubus ulmifolius [65]
52	Anthocyanin	Peonidin 3-O-(6-O-p-coumaroyl) glucoside	C31H29O13	609.554		609	303	257; 153	229	Grape [62]; vines [63]
53	Anthocyanin	Malvidin 3-O-(6-O-p-caffeoyl) glucoside	C32H31O15	655.5795		655	331; 493	315; 270; 153	270	Grape [62]; Bougainvillea [61]
54	Phenolic acid	Coniferyl aldehyde [4-Hydroxy-3-methoxycinnamaldehyde; Coniferaldehyde; Ferulaldehyde]	C10H10O3	178.1846		179	161; 133; 119	119		Juglans mandshurica [48]; potato [50]; A. cordifolia [53]
55	Phenolic acid	Caffeic acid derivative	C16H18O9Na	377.2985	377		341	215		Bougainvillea [61]; Embelia [54]
56	Hydroxybenzoic acid (Phenolic acid)	Ellagic acid [Benzoaric acid; Elagostasine; Lagistase; Eleagic acid]	C14H6O8	302.1926		303	257; 229; 165	229; 201	201	Juglans mandshurica [48]; Rhus coriaria [66]; Eucalyptus [87]
57	Phenylpropanoid (cinnamic alcohol glycoside)	Vimalin *	C16H22O7	326.3417		327	309; 195	241; 195		Rhodiola rosea [96]
58	Coumarin	Fraxetin *	C10H8O5	208.1675		209	167			Embelia [54]; Jatropha [47]; Artemisia martjanovii [97]
59	Lignan	Syringaresinol *	C22H26O8	418.4436		419	326; 253; 184	298; 254; 174	252; 226; 182	Wheat [98]; Annona montana [99]; Lonicera caerulea [79]
		Others
60		2,3-Dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one [DDMP]	C6H8O4	144.1253		145	127			Radix polygoni multiflori [56]
61	Aliphatic amino acid	L-Glutamic acid [L-Glutamate]	C5H7NO4	145.1134		146	144; 118			Medicago truncatula [100]; soybean leaves [101]; Lonicera japonica [45]
62	Amino acid	L-Histidine	C6H9N3O2	155.1546		156	110			Medicago truncatula [100]; Lonicera japonica [45]; Camellia kucha [80]; Actinidia deliciosa [102]; Lonicera caerulea [79]
63	Amino acid	Phenylalanine [L-Phenylalanine]	C9H11NO2	165.1891		166	120			Medicago truncatula [100]; Juglans mandshurica [48]; soybean [58]; soybean leaves [101]; Lonicera japonica [45]; potato leaves [92]; Camellia kucha [80]
64	Cyclohexenecarboxylic acid	Shikimic acid [L-Schikimic acid]	C7H10O5	174.1513		175	157			Medicago truncatula [100]; soybean [58]; Camellia kucha [80]; Ribes meyeri [44]
65	Tricarboxylic acid	cis-Aconitic acid	C6H6O6	174.1082		175	157			Medicago truncatula [100]
66	Tricarboxylic acid	Trans-Aconitic acid [trans-Aconitate]	C6H6O6	174.1082		175	157			Medicago truncatula [100]; Inula graveolens [41]
67	Amino acid	L-theanine [Theanine; Theanin; N-Ethyl-L-glutamine]	C7H14N2O3	174.1977		175	157			Camellia kucha [80]
68	Aromatic amino acid	Tyrosine [(2S)-2-Amino-3-(4-Hydroxyphnyl)Propanoic acid]	C9H11NO3	181.1885		182	177; 165	123		Medicago truncatula [100]; soybean leaves [101]; Hylocereus polyrhizus [103]; Polygala sibirica [104]
69	Essential amino acid	L-Tryptophan [Tryptophan; (S)-Tryptophan]	C11H12N2O2	204.2252		205	187	121		Camellia kucha [80]; Rosa acicularis [105]
70	Carboxylic acid	Myristoleic acid [Cis-9-Tetradecanoic acid]	C14H26O2	226.3550		227	209; 165	121		F. glaucescens [53]; Maackia amurensis [32];
71	Ribonucleoside composite of adenine (purine)	Adenosine	C10H13N5O4	267.2413		268	136	121		Lonicera japonica [45]; Huolisu oral liquid [30]; L. palustre [106]; Rosa acicularis [105]
72	Ribonucleoside composite of adenine (purine)	Inosine	C10H12N4O5	268.2261		269	136			Lonicera japonica [45]
73	Monosaccharides	6-Phosphogluconic acid	C6H13O10P	276.1352		277	259; 205; 188; 130			Medicago truncatula [100]
74	Omega-3 fatty acid	Linolenic acid (Alpha-Linolenic acid; Linolenate)	C18H30O2	278.4296		279	219; 154	159		Soybean [58]; soybean leaves [101]; Maackia amurense [32]; Polygala sibirica [104]
75	Polyunsaturated long-chain fatty acid	Hydroxy eicosatetraenoic acid	C20H32O3	320.4663		321	312; 256; 228; 193	224; 176; 143		F. glaucescens; F herrerae [53]
76	Polysaccharides	Adenosine 5′-monophosphate [5′-adenylic acid]	C10H14N5O7P	347.2212		348	341; 273; 233	205		Medicago truncatula [100]
77	Phytosterol	Ergosterol [Provitamin D2; Ergosterin]	C28H44O	396.6484		397	392; 311; 183; 129	361; 311; 226; 183; 130		F. glaucescens [53]
78	Pentacyclic triterpenoid	Sophoradiol	C30H50O2	442.7168		443	425; 175	175	115	Medicago truncatula [107]
79	Polysaccharides	Guanosine 5′-diphosphate	C10H15N5O11P2	443.2005		444	359; 323; 297; 274	189; 154	172	Medicago truncatula [100]
80	Anabolic steroid	Vebonol	C30H44O3	452.6686		453	435; 336; 209	336; 226	209	Rhus coriaria [66]; Hylosereus polyrhizus [103]
81	Triterpenic acid	Ursolic acid	C30H48O3	456.7003		457	411; 203	393; 283; 201	201	Juglans mandshurica [48]; Ocimum [34]; Mentha [36]
82	Triterpenic acid	Oleanolic acid	C30H48O3	456.7004		457	411; 263; 203	393; 309; 177	375; 203; 145	Medicago truncatula [107]; C. edulis [53]; Folium Eriobotryae [108]
83	Saponin	Soyasapogenol B [24-Hydroxysophoradiol; Soyasapogenin B]	C30H50O3	458.7162		459	452; 317; 279; 212	445; 298; 216		Medicago truncatula [107]
84	Saponin	Soyasapogenol A	C30H50O4	474.5434		475	437; 343; 249	168; 127		Medicago truncatula [107]
85	Polysaccharides	UTP [Uridine 5′-triphosphate]	C9H15N2O15P3	484.1411		485	478; 365; 321; 261			Medicago truncatula [100]
86	Polysaccharides	Guanosine-5′-triphosphate [GTP]	C10H16N5O14P3	523.1804		524	494; 386; 303; 165			Medicago truncatula [100]
87	Polysaccharides	UDP-arabinose [Uridine 5′-diphosphate arabinose]	C14H20N2O16P2	534.2599		535	275; 217; 159	220		Medicago truncatula [100]
88	Polysaccharides	Uridine diphosphate-xylose [UDP-xylose]	C14H22N2O16P2	536.2758		537	277; 187	249; 136		Medicago truncatula [100]
89	Polysaccharides	UDP-glucose [Uridine diphosphate glucose]	C15H24N2O17P2	566.3018		567	423; 385; 324; 283	405; 367; 297; 241		Medicago truncatula [100]
90	Product of chlorophyll degradation	Chlorophyllide a	C35H34MgN4O5	614.9733		615	583	565; 458; 269	520; 441	[109,110]
91		Medicagenic acid -3-O-beta-D-glucopyranoside	C36H56O11	664.8232		665	635; 510; 452; 401	337	319	Pubchem
92	Saponin	Azukisaponin II	C42H68O14	796.9809		797	519	429; 357; 243		Leguminosae [111]; Glycine max [112]
93	Saponin	Soyasaponin Bb’ [Soyasaponin III] *	C42H68O14	796.9809		797	519	429; 357; 243		Black soja [57]
94	Product of chlorophyll degradation	Pyropheophytin a	C53H72N4O3	813.1638		813	535	435; 329		[110]
95	Steroidal alkaloid	Alpha-chaconine *	C45H73NO14	852.0594		852	706; 560; 398	560	398	Potato [113,114]
96	Product of chlorophyll degradation	Pheophytin A	C55H74N4O5	871.1999		871	533	461		Physalis peruviana [115]; Capsicum [116]; [109, 110]
97	Saponin	Soyasaponin II [Soyasaponin II (SH); Soyasaponin Bc] *	C47H76O17	913.0961	912		501	483; 425	425	Black soja [57]; Leguminosae [111]; soya [117]
98	Saponin	Soyasaponin gamma g *	C48H74O17	923.0910		924	581; 423; 321	213		Black soja [57]; Leguminosae [111]; soya [117]
99	Saponin	3-Rhamnose-galactose-glucuronic acid-soyasapogenol B	C48H78O18	943.1221		944	598; 423; 295	581; 419; 215	572	Medicago truncatula [100]; Rhus coriaria [66]
100	Saponin	Soyasaponin I [Soyasaponin Bb] *	C48H78O18	943.1221		944	598; 423; 365; 281	205		Leguminosae [111]; soya [117]; Black soja [57];
101	Saponin	Soyasaponin Bd *	C48H76O19	957.1056		958	456; 595; 718; 812	409; 247	271	Black soja [57]; Leguminosae [111]; soya [117]
102	Saponin	6-deoxyhexose-hexoside-uronic acid-aglycone D	C48H78O20	975.1209		975	799; 715; 529; 477	301	286; 259; 201	Medicago truncatula [118]
103	Saponin	Soyasaponin beta g *	C54H84O21	1069.2322	1068		967; 879; 741; 584	659; 483		Black soja [57]; Leguminosae [111]; soya [117]

* Compounds identified for the first time in M. varia.

).

4. Discussion

The biologically active compounds of aerial parts of plants are effectively extracted using organic solvents such as methanol and ethanol. But extraction products in the final phase require additional purification from trace amounts of used solvents. SC-CO₂ extraction can be used as an alternative to traditional extraction methods: maceration or Soxhlet extraction [119,120]. SC-CO₂ extraction has been used in the evaluation of food products, the isolation of bioactive substances, and the determination of lipid levels in foods and levels of toxic substances. With SC-CO₂ extraction, the products do not contain organic solvent residues that occur with conventional extraction methods, and the solvents can be toxic, as in the case of methanol and n-hexane, for example. Easy solvent removal from the final product, high selectivity and moderate extraction temperatures are the main attractions of SC-CO₂ technology, leading to a significant increase in research for applications in the food and pharmaceutical industries. When comparing possible supercritical solvents, carbon dioxide has the most attractive advantages, being a non-toxic, non-flammable, environmentally friendly and renewable resource [121]. Popova et al. investigated the influence of SC-CO₂ extraction parameters and the quality of Ledum palustre feedstock on the global yields of chlorophylls and carotenoids. The data obtained were significant for the pharmaceutical, food, and perfume and cosmetic industries, which require natural dyes and antioxidants [122]. Baananou et al. reported the anti-inflammatory activity of two extracts from the aerial parts of Rhododendron [123]. Aliev et al., in their research, have shown that SC-CO₂ extraction is an effective method for extracting a wide range of lipophilic fractions from plant materials in one experimental procedure, which provides additional opportunities for research [124].

Thus, the use of SC-CO₂ extraction is an effective approach for the extraction of bioactive compounds. Our results are consistent with these reports that SC-CO₂ extraction is a useful approach to extract and study bioactive compounds.

The extracts obtained showed both a high content of polyphenolic compounds and a high content of saponin group compounds. Earlier studies revealed the presence and detection of polyphenols in legume species. Chiriac et al. [125] used UHPLC-Q exactive hybrid quadrupole orbitrap high-resolution mass spectrometry and identified 29 compounds from the sprouts of Medicago sativa and Trifolium pratense, based on their mass, FIs, retention time and data in the literature. However, using SC-CO₂ extraction, the number of polyphenol compounds was higher in our results. This difference could be due to the extraction method or the different tissues under study. Other than Medicago, polyphenols are also abundant in other legume species, such as Phaseolus vulgaris [126], soybean [127], chickpea, grass pea, lentils [128], peanut [129], etc. These polyphenols act as dietary antioxidants in humans and impart protective effects against certain diseases [130].

Apart from polyphenols, the presence of saponins in Medicago varieties is a useful observation. These observations are consistent with earlier studies which reported the presence of saponins in Medicago truncatula [100,107,118]. Saponins are active compounds present in edible legumes and impart health benefits [131,132]. Bioinformatic analyses in legumes have revealed the presence of the triterpene biosynthesis pathway and conserved genes [133,134]. Thus, the detection of eleven compounds classified as saponins and two triterpenoid acids (ursolic acid and oleanolic acid) in the leaves of M. varia is consistent with the above-cited studies. Though several studies on soybean and other legumes have highlighted that seeds and roots [135] are the major sinks for saponins, their presence in leaves and stems has also been reported in, e.g., Jatropha curcas [136], Acanthopanax sieboldianus [137], Quillaja lancifolia [138], etc. The detection of saponins in the leaves of M. varia together with a range of polyphenols suggests this plant as a potential raw material for use in traditional medicine as well as modern pharmacology.

It is well recognized that using some fungicides can be harmful to both the environment and human health. As a result, during the past twenty years, an increasing amount of research has been carried out on the potential use of plant-based substances that would be less harmful than chemicals produced in factories. The increasing prospect of employing saponins as natural fungicides is highlighted in a large number of international articles [139].

Research on 29 Medicago species has shown reliable results, including the identification of several species as having a high concentration of fungicidal saponins [140]. The saponins of some species, including M. sativa [141,142], M. arabica [143], M. arborea [144] and M. hybrida [145], have fungicidal actions.

Fourteen triterpene saponins from the roots of Medicago hybrida have been identified and their structures have been established [145]. Six fungi were tested in vitro to determine the antifungal activity of the roots’ saponins, and eight main saponin glycosides were tested against Botrytis tulipae, one of the most sensitive fungi [20].

It should be noted that different concentrations of saponins equally inhibited the mycelial growth of Botrytis cinerea and B. tulipae. However, the higher concentrations inhibited the mycelial growth of B. cinerea somewhat less. It is assumed that lower concentrations of saponins are sufficient to block all active sites in the mycelial hyphae. It has also recently been shown that M. hybrida saponins have insecticidal activities as high as those of M. arabica and Medicago murex [146]. On the other hand, it is known that M. arabica and M. murex saponins are also rich in highly fungicidal saponins [147]. In conclusion, Medicago saponins have significant antifungal activity, and the roots of this plant can be a rich source of natural fungicides. Therefore, their detection in the aerial parts of M. varia has added to the existing list of beneficial compounds.

5. Conclusions

M. varia species contain many polyphenolic components and components of other chemical groups that have valuable biological activities. SC-CO₂ extraction of M. varia (three varieties) was successfully carried out by the team of authors. Certain extraction conditions were selected, and the extracts obtained showed both a high content of polyphenolic compounds and a high content of saponin group compounds. Tandem mass spectrometry (HPLC-ESI–ion trap) was used to detect target analytes. Mass spectrometric data were recorded on an ion trap equipped with an ESI source in negative and positive ion modes. A four-stage ion separation mode was implemented. One hundred and three different biologically active compounds were found in M. varia extracts. Twenty-seven phenolic compounds were tentatively identified for the first time. Also, for the first time, the following saponins were identified with reliable accuracy in M. varia: soyasaponin II, soyasaponin gamma g, soyasaponin I, soyasaponin Bd, soyasaponin beta g, steroidal alkaloids (alpha-chaconine), etc. Although our study aimed only to study the aerial parts of Medicago varia, which belongs to the legume family (Fabaceae), the same approach can be applied in the future to study factors influencing the metabolite profiles of legume seeds, including seasonal variations, cultivation and storage conditions. In addition, our study may provide new interesting details for future taxonomic studies, especially if they target larger genotypes.